Abstract

A thermostable arginase (l-arginine amidinohydrolase, EC 3.5.3.1) was purified from the extreme thermophile ‘Bacillus caldovelox’ (DSM 411) by a procedure including DEAE-Sepharose chromatography, and gel filtration, anion exchange and hydrophobic-interaction fast-protein liquid chromatography, with substantial retention of the metal ion cofactor. The purified enzyme is a hexamer with a subunit Mr of 31000 ± 2000 and contains ≥1 Mn atom per subunit. Maximum activation on incubation with Mn²⁺ is 29%. Activity is optimal at pH 9 and at 60°C the Km for arginine is 3.4 mM and Ki(ornithine) is 0.55 mM. Incubation in 0.1 M Mops/NaOH buffer (pH 7) causes rapid inactivation at 60°C (t1/2(half life) = 4.5 min) and individually 0.1 mM Mn²⁺ or 1 mg/ml BSA (bovine serum albumin) increase the t1/2 of arginase activity 4-fold, but combined they produce > 1000-fold increase and a t1/2 = 105 min at 95°C. Aspartic acid and other species that bind Mn²⁺ can replace BSA, and it is suggested that arginase can be activated by free Mn²⁺. A strong chelating agent causes inactivation without subunit dissociation, but arginase dissociates rapidly at pH 2.5. Reassociation occurs at pH 9 and is unusual in that it does not require Mn²⁺.